scholarly journals Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

2011 ◽  
Vol 77 (22) ◽  
pp. 8193-8196 ◽  
Author(s):  
Lucja M. Jarosz ◽  
Bastiaan P. Krom
Keyword(s):  
Candida Albicans ◽  
Real Time ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Rapid Screening ◽  
Content Type ◽  
Wide Range ◽  
Hyphal Formation ◽  
Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

scholarly journals Vibrio harveyi Adheres to and Penetrates Tissues of the European Abalone Haliotis tuberculata within the First Hours of Contact

2014 ◽  
Vol 80 (20) ◽  
pp. 6328-6333 ◽  
Author(s):  
Marion Cardinaud ◽  
Annaïck Barbou ◽  
Carole Capitaine ◽  
Adeline Bidault ◽  
Antoine Marie Dujon ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Vibrio Harveyi ◽  
Bacterial Pathogen ◽  
Content Type ◽  
Infection Dynamics ◽  
Haliotis Tuberculata ◽  
Green Fluorescent ◽  
Portal Of Entry

ABSTRACTVibrio harveyiis a marine bacterial pathogen responsible for episodic epidemics generally associated with massive mortalities in many marine organisms, including the European abaloneHaliotis tuberculata. The aim of this study was to identify the portal of entry and the dynamics of infection ofV. harveyiin the European abalone. The results indicate that the duration of contact betweenV. harveyiand the European abalone influences the mortality rate and precocity. Immediately after contact, the epithelial and mucosal area situated between the gills and the hypobranchial gland was colonized byV. harveyi. Real-time PCR analyses and culture quantification of a green fluorescent protein-tagged strain ofV. harveyiin abalone tissues revealed a high density of bacteria adhering to and then penetrating the whole gill-hypobranchial gland tissue after 1 h of contact.V. harveyiwas also detected in the hemolymph of a significant number of European abalones after 3 h of contact. In conclusion, this article shows that a TaqMan real-time PCR assay is a powerful and useful technique for the detection of a marine pathogen such asV. harveyiin mollusk tissue and for the study of its infection dynamics. Thus, we have revealed that the adhesion and then the penetration ofV. harveyiin European abalone organs begin in the first hours of contact. We also hypothesize that the portal of entry ofV. harveyiin the European abalone is the area situated between the gills and the hypobranchial gland.

scholarly journals Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples

2017 ◽  
Vol 83 (14) ◽  
Author(s):  
Kuppuswamy N. Kasturi ◽  
Tomas Drgon
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
False Negative ◽  
Screening Method ◽  
Reference Method ◽  
Rapid Screening ◽  
Content Type ◽  
Pcr Method ◽  
Group D

ABSTRACT The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA, group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non-Salmonella organisms. The invA- and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella-differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S. Enteritidis isolates and 100% exclusivity for the 297 non-Enteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the Vitek immunodiagnostic assay system (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method. IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples.

scholarly journals Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

2011 ◽  
Vol 55 (5) ◽  
pp. 2438-2441 ◽  
Author(s):  
Zeynep Baharoglu ◽  
Didier Mazel
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Vibrio Cholerae ◽  
Fluorescent Protein ◽  
Resistance Development ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Green Fluorescent ◽  
Regulated Promoters

ABSTRACTAntibiotic resistance development has been linked to the bacterial SOS stress response. InEscherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS inVibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation withE. coli, all these antibiotics induce SOS inV. cholerae.

scholarly journals New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

2016 ◽  
Vol 82 (8) ◽  
pp. 2240-2246 ◽  
Author(s):  
Alex I. Kanno ◽  
Cibelly Goulart ◽  
Henrique K. Rofatto ◽  
Sergio C. Oliveira ◽  
Luciana C. C. Leite ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Expression Vectors ◽  
Content Type ◽  
Expression Levels ◽  
Wide Range ◽  
Mycobacteriophage L5 ◽  
Green Fluorescent ◽  
Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

scholarly journals Identification and Characterization of Rvs162/Rvs167-3, a Novel N-BAR Heterodimer in the Human Fungal Pathogen Candida albicans

2014 ◽  
Vol 14 (2) ◽  
pp. 182-193 ◽  
Author(s):  
Areti Gkourtsa ◽  
Janny van den Burg ◽  
Karin Strijbis ◽  
Teja Avula ◽  
Sietske Bijvoets ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fluorescent Protein ◽  
Genetic Interaction ◽  
Phenotypic Analysis ◽  
Content Type ◽  
Associated Functions ◽  
Increased Sensitivity ◽  
Green Fluorescent ◽  
And Function

ABSTRACT Membrane reshaping resides at the core of many important cellular processes, and among its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro . A second, distinct heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes, indicating that C. albicans expresses two functional BAR heterodimers. We used live-cell imaging to localize green fluorescent protein (GFP)-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly colocalizes with the endocytic marker protein Abp1, we do not observe any colocalization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3 Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt concentrations or decreased cell wall integrity, phenotypes which have been observed for rvs167 Δ/Δ and rvs161 Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans : the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles, our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have related but distinct membrane-associated functions.

scholarly journals TheRTA3Gene, Encoding a Putative Lipid Translocase, Influences the Susceptibility of Candida albicans to Fluconazole

2016 ◽  
Vol 60 (10) ◽  
pp. 6060-6066 ◽  
Author(s):  
Sarah G. Whaley ◽  
Sarah Tsao ◽  
Sandra Weber ◽  
Qing Zhang ◽  
Katherine S. Barker ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Clinical Isolate ◽  
Fluorescent Protein ◽  
Microtiter Plate ◽  
Microtiter Plate Assay ◽  
Content Type ◽  
Activating Mutations ◽  
Gene Coding ◽  
Activating Mutation ◽  
Green Fluorescent

ABSTRACTTheRTA3gene, coding for a member of the Rta1p-like lipid-translocating exporter family, is coordinately upregulated with the ATP-binding cassette transporter genesCDR1andCDR2in azole-resistant clinical isolates ofCandida albicansthat carry activating mutations in the transcription factor Tac1p. We show here that deletingRTA3in an azole-resistant clinical isolate carrying a Tac1p-activating mutation lowered fluconazole resistance by 2-fold, while overexpressingRTA3in an azole-susceptible clinical isolate resulted in enhanced fluconazole tolerance associated with trailing growth in a liquid microtiter plate assay. We also demonstrate that an Rta3p-green fluorescent protein (GFP) fusion protein localizes predominantly to the plasma membrane, consistent with a putative function for Rta3p as a lipid translocase.

scholarly journals Motor Protein Myo5p Is Required To Maintain the Regulatory Circuit ControllingWOR1Expression in Candida albicans

2012 ◽  
Vol 11 (5) ◽  
pp. 626-637 ◽  
Author(s):  
Nadezda Kachurina ◽  
Bernard Turcotte ◽  
Malcolm Whiteway
Keyword(s):  
Candida Albicans ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Content Type ◽  
Bud Emergence ◽  
Regulatory Circuit ◽  
Myosin I ◽  
Bud Growth ◽  
Green Fluorescent ◽  
Germ Tubes

ABSTRACTTheCandida albicans MYO5gene encodes myosin I, a protein required for the formation of germ tubes and true hyphae. Because the polarized growth of opaque-phase cells in response to pheromone results in mating projections that can resemble germ tubes, we examined the role of Myo5p in this process. We localized green fluorescent protein (GFP)-tagged Myo5p in opaque-phase cells ofC. albicansduring both bud and shmoo formation. In vegetatively growing opaque cells, Myo5p is found at sites of bud emergence and bud growth, while in pheromone-stimulated cells, Myo5p localizes at the growing tips of shmoos. Intriguingly, cells homozygous forMTLain which theMYO5gene was deleted failed to switch efficiently from the white phase to the opaque phase, although ectopic expression ofWOR1from theMET3promoter can convertmyo5mutants into mating-competent opaque cells. However, whenWOR1expression was shut off, themyo5-defective cells rapidly lost both their opaque phenotype and mating competence, suggesting that Myo5p is involved in the maintenance of the opaque state. WhenMYO5is expressed conditionally in opaque cells, the opaque phenotype, as well as the mating ability of the cells, becomes unstable under repressive conditions, and quantitative real-time PCR demonstrated that the shutoff ofMYO5expression correlates with a dramatic reduction inWOR1expression. It appears that while myosin I is not directly required for mating inC. albicans, it is involved inWOR1expression and the white-opaque transition and thus is indirectly implicated in mating.

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